Effects of Ractopamine Hydrochloride on Color Attributes and Meat Quality in Yearling Heifers Across Days on Feed. Erin Nicole Hunter

Transcription

1 Effects of Ractopamine Hydrochloride on Color Attributes and Meat Quality in Yearling Heifers Across Days on Feed by Erin Nicole Hunter A thesis submitted to the Graduate Faculty of Auburn University in partial fulfillment of the requirements for the Degree of Master of Science Auburn, Alabama May 14, 2010 Keywords: Ractopamine hydrochloride, fiber types, color, tenderness Copyright 2010 by Erin Nicole Hunter Approved by Chris Kerth, Chair, Professor of Animal Sciences Christy Bratcher, Assistant Professor of Animal Sciences Lisa Kriese-Anderson, Associate Professor of Animal Sciences Terry Brandebourg, Assistant Professor of Animal Sciences

2 Abstract Recent research with the use of β-agonists has been inconclusive from a meat quality standpoint. Therefore, the objective of this study is to evaluate the inclusion of RAC across 5 DOF in 72 crossbred yearling heifers and how it effects meat quality. Heifers were individually fed twice a day at the Auburn University Beef Cattle Evaluation facility. Heifers were divided based on initial height and weight, and randomly assigned to one of the following DOF groups: 79 (n = 16), 100 (n = 16), 121 (n = 16), 142 (n = 16), or 163 (n = 7). Within these 5 groups, heifers were randomly assigned to a treatment of either 300 mg hd -1 d -1 RAC-HCl (RAC; Elanco Animal Health, Greenfield, IN) for the final 35 d prior to harvest or a control group (CON; 0 mg hd -1 d -1 RAC). Twenty-four hours postmortem, boneless loins were removed from the right side of each animal, vacuum-packaged, and aged for 21 d in the cooler (2 o C). After 21 d, 7 strip steaks (2.54 cm thick) were cut for sensory analysis, WBSF, and lipid oxidation analysis. The main effect of RAC had no effect (P > 0.05) on meat quality. Hot carcass weight was lowest (P < 0.05) for DOF group 79 compared to other DOF treatment groups. Backfat thickness and KPH was highest (P < 0.05) in DOF groups 121 and 142. Intermediate fiber area was highest (P < 0.05) for DOF group 121. The percentage of red fibers was highest (P < 0.05) for DOF group 121. Compared to all other treatments, DOF group 163 RAC steaks were less tender for initial and sustained tenderness. RAC supplementation had no effect on carcass characteristics and there were no deleterious effects on meat quality factors by the addition of RAC into the diet. ii

3 Acknowledgments There are so many people that have contributed to the completion of this project. First of all, I would like to thank my major professor, Dr. Chris Kerth, for the opportunity to obtain a Master s degree under his direction in Meat Science and Muscle Biology. He has provided me with a wide knowledge of the field as well as experiences that will never be forgotten. I would also like to thank my committee members, Dr. Christy Bratcher, Dr. Terry Brandebourg, and Dr. Lisa Kriese-anderson for direction on this project. My fellow graduate students Clint Rowe, Katie McMurtrie and Danny Johns have also offered help and support whenever needed. There has also been plenty of learning and good times at the meat lab with all of the workers. A special thanks to Barney Wilborn, Pete Holloway, and T.J. Mayfield for help and laughs. My parents have always been the most supportive people in my life and have been behind me since I began my journey at Auburn University. I also would like to thank my best friend, roommate and fellow graduate student Kim Cline for always being there the last six years (hard to believe). Finally, my boyfriend Chad Beasley has watched me progress through both undergraduate and graduate school and has always been there for me with love and support, thank you! iii

4 Table of Contents Abstract... ii Acknowledgments... iii List of Tables and Figures... vii I. Review of Literature... 1 Introduction... 1 Muscle Structure... 2 Skeletal Muscle Growth... 4 Postmortem Degradation and Meat Tenderness... 6 β-adrenergic Agonists... 8 Dosage, Length of Supplementation, and Sex Effects Carcass Characteristics and Days on Feed β-agonists and Muscle Growth Warner-Bratzler Shear Force and Sensory Characteristics of cattle finished on β-agonists Meat Color Lipid Oxidation Summary iv

5 II. Materials and Methods Animal Care Carcass Evaluation Sample Preparation Color Measurements Warner-Bratzler Shear Force Trained Sensory Evaluation Histochemical Fiber Typing TBARS Statistical Design III. Results Carcass Traits Muscle Fiber Types Color TBARS WBSF Sensory Evaluation IV. Discussion Carcass Traits Muscle Fiber Types Color TBARS WBSF v

6 Sensory Evaluation Implications V. Tables and Figures Literature Cited Appendices vi

7 List of Tables and Figures Table 1. Diet Composition fed to yearling heifers...37 Table 2. Least squares means of carcass characteristics for heifers as influenced by RAC across five days on feed...38 Table 3: Table 2. Least squares means of carcass characteristics for heifers as influenced by RAC across five days on feed...39 Table 4. Least squares means of color measurements as affected by RAC across five days on feed...40 Table 5. Least squares means of sensory characteristics for RAC and CON steaks across days on feed...41 Figure 1. Least squares means of marbling scores in yearling heifers as affected by RAC across five days on feed...42 Figure 2. Least squares means of 630/580 ratio of steaks displayed for 7 d as influenced by RAC, days on feed, and display day...43 Figure 3. Least squares means of saturation index across DOF in a seven day retail display time Figure 4. Least squares means of hue angle across DOF in a seven day retail display time...45 Figure 5. Least squares means of a* values across DOF in a seven day retail display time...46 Figure 6. Least squares means of b* values across DOF in a seven day retail display time...47 Figure 7. Least squares means of Thiobarbituric Acid Reactive Substances (TBARS) of steaks displayed and not display...48 Figure 8. Least squares means of flavor intensity for steaks across five days on feed...49 Figure 9. Least squares means of off-flavor of steaks not in display and steaks after a 7 day retail display time...50 vii

8 Figure 10. Least squares means of Warner Bratzler Shear Force (WBSF) for steaks influenced by 7 day retail display time viii

9 Introduction I. LITERATURE REVIEW In today s market, increasing input costs for producers have directed research efforts towards finding more cost-effective production options. Improved methods for decreasing the feed required per unit of gain in live animals are essential to alleviate input costs wherever possible. In order to decrease the amount of feed consumed in an animal s life, certain repartitioning agents, known as β-agonists, have been developed to increase average daily gain in the animal, increase lean muscle accretion, and decrease fat accretion (Mersmann, 1998). Because of this increase in ADG, meat animals reach desired harvest weights in a shorter amount of time. At the same time, a decrease in subcutaneous fat and increase in lean muscle should result in lower USDA yield grades resulting in an overall increase in profit. Data published in the early 1980 s reported data published by the Cyanamid company which revealed that oral administration of clenbuterol, a β-adrenergic agonist, increased muscle mass and decreased fat mass in growing cattle, pigs, chickens, and sheep (Ricks et al., 1984). In more recent years, β-adrenergic agonists such as Ractopamine hydrochloride (RAC), which was the first β-agonist approved by the FDA in 2003 for use in cattle, have been implemented into the production system. These β-agonists tend to increase lean muscle, through muscle hypertrophy, and decrease tenderness, possibly due to increased levels of proteolytic inhibitors and decreased protein degradation. This review will cover the published literature related to the relationship between β-agonists and their effects on skeletal muscle growth, tenderness, shelf-life stability, and consumer acceptance. 1

10 Muscle Structure There are two main protein components which make up the muscle, consisting of the muscle and connective tissue. Within these two compartments are three classes of proteins as described by Goll et al. (2008). First, the stroma proteins make up approximately 10-15% of total protein and can be classified as those that make up the connective tissue system, with the major contributor being the fibrous protein, collagen. The sarcoplasmic proteins constitute approximately 30-35% of the total protein in the muscle, are soluble in water, and are typically enzymes involved in the metabolic pathway, creatine kinase, and myoglobin (Tornberg, 2005). The majority of muscle protein is represented by myofibrillar proteins (55-60%), which includes the major muscle proteins actin and myosin (Goll et al., 2008). The myofibrillar proteins are responsible for the contractile properties of the muscle and therefore can have adverse effects on meat quality depending on the mechanisms in the muscle. The myofibrillar proteins are the majority of the total protein content in the muscle and are located within the myofibril. This group is composed of a vast number of proteins, which are all involved in processes related to muscle structure, and more specifically sarcomere structure. The sarcomere is the basic contractile unit in the myofibril. The muscle fiber is made up of many proteins, which serve as both regulatory and structural proteins. These proteins range from those found in the Z-disk of the sarcomere to those that are associated with the structure of the myofiber. Within the sarcomere are two myofibrillar proteins known as myosin and actin, which are components of the thick and thin filaments, respectively. Aside from the proteins in the lean muscle, connective tissue is also a large component within the animal. Collagen is the main protein of connective tissue in muscle. It is the most abundant mammalian protein, providing a network of fibers in all tissues (McCormick, 1999). 2

11 Presently, 19 known forms of collagen have been identified, which hold roles in a variety of biological systems (McCormick, 1999). The structure of various collagen forms differ from mesh-like networks of Type IV collagen to long, filamentous fibers typical of Types I, II, and III. The basic structure of connective tissue in muscle includes three compartments known as the epimysium, perimysium, and endomysium. The epimysium surrounds an individual muscle and is continuous with the tendon joining other muscles or bones in the epimysium (McCormick, 1999). Due to its support role, the epimysium is often very thick, and is resistant to both heat and force. Although very tough, it generally does not affect meat quality because it is typically separated from meat in order to avoid decreases in quality. The perimysium is responsible for covering muscle bundles, or fascicles, and is predominantly composed of collagen types I & III (Purslow, 2005). Along with surrounding muscle bundles, the perimysium also contains intramuscular lipid deposits and vasculature. There are large or primary fascicles and small or secondary fascicles, and therefore primary and secondary perimysial layers separate them (Purslow, 2005). Finally, the endomysium is the innermost constituent of the collagen compartments, which encompasses the individual muscle fibers and overlays the basement membrane. Although, the intramuscular connective tissue is a combination of both the peri- and endomysiums, the perimysium constitutes about 90% of the tissue (McCormick, 1999). Therefore, the perimysium is a major determinant of meat quality and more specifically, meat tenderness (Purslow, 1999). One way that the perimysium affects meat quality is that its thickness increases as an animal ages (Brooks and Savell, 2004) 3

12 Skeletal Muscle Growth Current research is focused on improving methods for producing more efficient, lean animals. These characteristics can be achieved by a number of factors associated with muscle growth including an increase in muscle fiber number, fiber size, and a shift from small, oxidative slow-twitch fibers to large, glycolytic fast-twitch fibers (Wegner et al., 2000). There are also studies suggesting that fiber type and size has an impact on overall eating quality, including tenderness (Crouse et al., 1991; Maltin et al., 1998), but this relationship still remains unclear. Other studies, including Whipple et al. (1990) and Vestergaard et al. (2000) concluded that there was no correlation between fiber type and tenderness. Muscle growth is a result of the increase in number of muscle fibers (hyperplasia) and growth of the individual fiber (hypertrophy). Muscle fibers are formed during the embryonic and fetal stages of life prior to birth. The number of muscle fibers is thought to be fixed at birth in most mammals (Andersen, 2005). Therefore, postnatal growth is the increase in crosssectional area and the addition of sarcomeres contributing to the length of the muscle fibers. Muscle development of an animal is a combination of both hyperplasia and hypertrophy both in prenatal and postnatal development. However, hypertrophy is determined by both intrinsic and extrinsic factors, and occurs as the animal ages (Koohmaraie et al., 2002). Intrinsic factors can be breed type and sex of the animal, whereas extrinsic factors may include feeding regime and environmental conditions that the animal is exposed to. The amount of muscle growth is determined by the amount of protein synthesis as well as the protein degradation (Koohmaraie et al., 2002). The difference in these two factors is the determination of overall protein accretion. This process can be affected by three possible scenarios; a) increased protein synthesis and decreased protein degradation, b) protein synthesis in amounts greater than the amount of protein 4

13 degradation, c) protein synthesis in amounts lesser than in protein degradation (Koohmaraie et al., 2002). All of these factors can have both positive and negative results on meat tenderness. Muscle fiber types are characterized by their metabolic capabilities, either oxidative or glycolytic metabolism (Klont et al., 1998). The type I fibers, also known as β-red fibers, are slow-twitch and exhibit oxidative metabolism. These fibers appear red after staining due to the number of mitochondria in the cell. Type IIa, or α-red fibers, are slow twitch and can metabolize both anaerobically or aerobically. Finally, the type IIb, or α-white fibers, are those that are fasttwitch, glycolytic fiber types. As previously stated, there are two types of α-fibers, which are either intermediate or white fiber types. Many studies conclude there is a transformation postnatally which determines whether a fiber will be intermediate or white (Ashmore, 1974). Following birth, the transformation of α fibers into either α-red or α-white is caused by the function of the muscle. Results of Ashmore (1974) suggested that muscles that are relatively dormant show a transformation from α-red to α-white, as well as a rapid growth in diameter. In contrast to α fibers, β fibers grow at a much slower rate and do not undergo a transformation. This theory also supports the idea that β fibers are located internally in the fiber bundles of the muscle, whereas α fibers are mostly seen on the peripheral areas of the muscle (Ashmore et al., 1972). Ashmore, Tompkins, and Doerr (1972) conducted an experiment using ovine, porcine, and bovine muscles to determine whether the transformation of α fibers occurred similarly in these species as it had in a previous experiment by a group of chicks. The current study again showed that the muscles from ovine, porcine, and bovine did undergo transformation of α fibers and the transformation appeared to be primarily concerned with energy-producing enzymes. As mitochondrial density decreased, glycogen phosphorylase increased. Glycogen phosphorylase is 5

14 indicative of glycogen enzymes in general and is an indicator of anaerobic or glycolytic metabolic processes. Examining the typical growth curve is important when conducting studies which affect the animal s nutrition, and thus, affecting animal growth. As changes are made in the diet, whether it is through the use of β-agonists or growth promotants, it is important to acknowledge they will undoubtedly affect the animal s growth, and can cause a shift in fiber types. Vestergaard et al. (1994) stated that β-agonists increase the percentage of type IIA fibers at the expense of type I fibers. Postmortem Degradation and Meat Tenderness Meat tenderization is achieved through a number of processes and can be affected by many factors. The end product of meat tenderization is the breakdown of myofibrillar proteins and the ultimate degradation of the Z-line in the sarcomere (Koohmaraie, 2002). In order for these processes to occur there is an enzyme system which is involved in protein degradation known as the calpain system (Koohmaraie, 2002). Calpains are calcium-dependent enzymes that bind to calcium, allowing the enzymes to breakdown proteins. The two basic calpain types are known as µ- and m-calpain, which differ in the amount of calcium required to activate the enzyme. Therefore, µ-calpain is activated by very low levels of calcium, while m-calpain requires higher levels. Because of their role in the breakdown of proteins, the calpains are thought to be the rate-limiting step in postmortem proteolysis (Goll et al., 2008). The known inhibitor of the calpains is calpastatin. Many studies have attributed decreased tenderness to increased levels of calpastatin in meat products because of their inhibition of the calpain system in proteolysis (Koohmaraie et al., 1991; Morgan et al., 1993) 6

15 Postmortem proteolysis results in degradation of the structural components of the sarcomere, and more specifically the Z-disk (Koohmaraie, 2002). The Z-disk is comprised of many proteins which enhance the structural integrity of the sarcomere. In order for proteolysis to degrade the proteins of the Z-disk, the calcium-dependent calpains must be activated. Several studies have shown degradation of the Z-disk begins within the first h postmortem, and degradation continues for d. Wheeler and Koohmaraie (1994), illustrated there is a large increase in shear value from 1-12 h (5.07 kg to 8.24 kg) postmortem. Following this increase in shear force values, there was a steady decline between 12 and 72 h postmortem, in which shear values decreased from 8.24 kg to 4.36 kg. Following 72 h., shear force decreased at a decreasing rate. By d 14 postmortem, shear force values had decreased to an average of 3.10 kg. Along with myofibrillar tenderness, connective tissue tenderness is one of the major determinants in overall meat tenderness and accounts for background tenderness in the product (Bailey, 1972). There are many factors affecting the amount of collagen, as well as collagens affect on meat tenderness. These factors can range from live animal factors, such as age, to factors associated with meat cookery (Purslow, 2005). Collagen has been shown to decrease the tenderness of meat because of the formation of cross-links in the muscle (Nishimura et al., 1995). This cross-link formation is often a result of animal age. Many studies have shown the total collagen content increases as an animal ages, which results in the replacement of reducible crosslinks with mature forms (hydroxyproline crosslinks), because of a decrease in the rate of collagen turnover (McCormick, 1999; Harper et al., 1999; Purslow, 2005). These crosslinks are assembled by formation of covalent bonds, which replace hydrogen bonds. As total collagen content increases, there is also a decrease in soluble collagen in muscles from older animals, which results in higher amounts of insoluble 7

16 collagen. Therefore, the muscle may suffer from tenderness issues because cooking temperatures will not breakdown insoluble collagen. With the understanding that animal age increases the number of mature crosslinks in the muscle, research efforts have focused to increase collagen turnover due to nutrition, resulting in newly synthesized collagen. In a study conducted by Boleman et al. (1996), mature cows were placed in feedlot conditions for 0, 28, 56, and 84 d prior to slaughter. Through the use of a highconcentrate diet, this study showed newly synthesized heat-soluble collagen tended to increase in the muscle. Another study by Aberle et al. (1981) showed an intensive preslaughter feeding regimen for 70 d, increased the percentage of soluble collagen as a result of increased rate of protein synthesis. Although age plays a major role in the formation of collagen, there are also other factors associated with increases in total collagen content. These factors include muscle and collagen type within the muscle. Locomotive muscles, such as the semimembranosus in the round, have shown to contain more collagen than support muscles such as the longissimus (Brooks and Savell, 2004). As previously mentioned, soluble collagen content within the muscle is susceptible to breakdown with heat and therefore does not cause tenderness issues unlike insoluble collagen does. β-adrenergic agonists Beta-agonists have been utilized for many years in research in beef cattle, chicken, sheep, and swine. Cunningham (1965) was the first to present data showing that growth could be changed by administering agents such as caffeine, theophylline, nicotine, and epinephrine. It was not until the 1980 s that data was published which indicated a model of growth for animals fed the β- agonist, clenbuterol, which increased muscle and decreased fat mass (Ricks et al., 1984). The β- 8

17 agonists are also referred to as repartitioning agents because they alter muscle tissue without effecting bone or organ mass (Beerman, 2002). Various β-agonists have different effects on animals due to factors such as species, duration of agonist feeding, dosage levels and sex effect. Williams (1989) suggested that the lack of response in very young nursing lambs and in other species to β-agonist treatment is due to an insufficient number of receptors or that growth is already at its peak rate in very young muscle fibers. Ractopamine hydrochloride (RAC; Elanco Animal Health; Greenfield, IN) was the first β-adrenergic agonist approved by the US Food and Drug Administration (FDA) for use in cattle finishing diets to increase rate of weight gain, improve feed efficiency and increase carcass leanness when fed the last 28 to 42 d (Schroeder et al., 2004a), prior to harvest. Ractopamine, which is a part of the phenethanolamine class of compounds, is responsible for binding to β- adrenergic receptors in order to elicit the β-agonist response in livestock species. For a β-agonist to have biological activity, it must have a substituted six-membered aromatic ring, hydroxyl group bonded to the β-carbon in the R configuration, positively charged nitrogen in the ethylamine side chain, and bulky substituent on the aliphatic nitrogen to allow specificity for the β-receptor (Weiner, 1980; Smith, 1998). Slight modification to the structure of β-agonists causes differences in biological activity. More specifically, structural changes seen on the aromatic ring groups and the bulky R-groups off of the aliphatic nitrogen, account for differential responses elicited by β-agonists (Mersmann et al., 1998). It is also known that the addition of halogens increase the lipophilicity of the aromatic portion of the β-agonist opposed to the hydroxylated aromatic ring (Smith, 1998). The structure of phenethanolamines is similar to that of catecholamines, which include the natural adrenergic neurotransmitters epinephrine and 9

18 norepinephrine that only differ in the bulky group on the aliphatic nitrogen. Therefore the mechanism for which a β-receptor binds to the agonist is similar. Mersmann (1998) reviewed the mechanism associated with the action of the β-adrenergic agonist and receptor relationship in order to stimulate a response of epinephrine and norepinephrine, which are known physiological β-adrenergic receptor agonists. This mechanism begins with the activation of the G s protein. This is achieved by the binding of the organic molecule, the β-adrenergic agonist, to a β-adrenergic receptor. These receptors are present in most mammalian cells, but the distribution of subtypes is dependent on tissue location and species (Mersmann, 1998). Once the G s protein is activated by the agonist-receptor complex, the α-subunit of the G s protein activates the enzyme adenylyl cyclase. This enzyme is responsible for the production of cyclic adenosine monophosphate (camp). This intracellular signaling molecule can then bind to the regulatory subunit of protein kinase A (PKA) to release the catalytic subunit to phosphorylate intracellular proteins. Once PKA is activated, a number of activities can be present. For example, many proteins can be activated when PKA is phosphorylated including hormone sensitive lipase, which is the rate-limiting enzyme for lipolysis (Mersmann, 1998). Also, enzymes such as acetyl-coa carboxylase, which is the ratelimiting enzyme for lipogenesis, are also inactivated due to phosphorylation. Finally, PKA can have an impact on gene expression by phosphorylating transcription factors. For example, gene expression can be activated or inactivated by the phosporylation of camp response element binding (CREB) protein (Mersmann, 1998). Due to the activation of receptors which have effects on enzymes involved in both lipogenesis and lipolysis, the final amount of fat deposited in the animal is affected by β-agonists. 10

19 Dosage, length of supplementation, and sex effects External factors such as dose, length of treatment, and sex effects, are also associated with the magnitude of the β-agonist response (Moody et al., 2000). Sainz et al. (1993) showed pigs fed 20 mg/kg RAC for 6 wk had an increase in average daily gain from week 0-3, but showed no improvement in the final 3 wk prior to slaughter. In a study conducted by Chikhou et al. (1993), steers were fed cimaterol from 4 wk of age until slaughter. These results concluded there was little effect on growth rate and gain efficiency in comparison to trials that fed β- agonists for a shorter amount of time. In contrast, Beerman et al. (1986, 1987), showed that lambs fed cimaterol for approximately 2 mo had a 25-30% increase in the weights of several muscles compared to those lambs on the control diet. Based on all of these studies, it is evident that β-agonists can have differing effects based on many factors. When evaluating the response of RAC in heifers compared to steers, studies have shown a decreased response in certain carcass characteristics. Schroeder et al. (2004b) reported no change in dressing percentage for heifers, whereas dressing percentage of steers in the same study had increased values when fed 200 or 300 mg hd -1 d -1 RAC. Additionally, ribeye area and carcass maturity were only improved in heifers fed the maximum dosage level approved by the FDA (300 mg. hd -1. d -1 ). Carcass characteristics and days on feed In respect to cattle, conflicting results for carcass characteristics in steers and heifers finished on RAC are found in the literature. Laudert et al. (2004) showed that yearling beef steers had increased average daily gain, improved feed efficiency, increased carcass weights, and limited effects on USDA yield and quality grades. In steers finished for the final 28 and 42 d on mg hd -1 d -1 RAC, Schroeder et al. (2004a) found heavier hot carcass weights, increased 11

20 ribeye area, increased dressing percent for 200 and 300 mg hd -1 d -1 fed steers, and higher yield grade in 300 mg hd -1 d -1 group. The same trial was conducted using heifers in which differences were found only in hot carcass weight and ribeye area. These results showed that hot carcass weights was increased in only the 200 and 300 mg hd -1 d -1 RAC groups and ribeye area was increased in heifers fed 300 mg hd -1 d -1 RAC. A similar trial on heifers concluded that there were no differences in hot carcass weight or ribeye area (Quinn et al., 2008). These results contrast the results by Schroeder et al. (2004a) as well as Gruber et al. (2007) in which these two studies found no differences in hot carcass weight and dressing percentage on heifers finished on 200 mg hd -1 d -1 RAC in the final 28 d of the finishing phase. Beta-agonist action in beef cattle is also affected by the number of days on feed. Previously, studies such as Winterholler et al. (2007) showed that yearling steers fed RAC increased hot carcass weight, dressing percentage, and ribeye area when time on feed increased. Marbling score and USDA yield grade both improved. These results were comparable with May et al. (1992) as well as Van Koevering et al. (1995) which concluded that an increase in days on feed would increase yield grade and subcutaneous fat cover. All of these projects show that increased days on feed, with or without RAC supplementation, reach a harvest optimum, which is consistent with the animal s growth curve. One negative effect of increasing days on feed, is that the maturity of the animal increases. As previously stated, animal age results in the development of greater amounts of insoluble collagen, resulting in a less tender product. Shackelford et al. (1995) reported differences in overall tenderness and ease of fragmentation between yearling heifers and 24 month-old cows. As early as 1970, Zinn et al. (1970) concluded that there was a significant 12

21 interaction between days on feed and tenderness. Cattle fed 180 days were significantly tougher than cattle fed for 120 to 150 d. Beta-agonists and muscle growth The β-agonists have shown to be the result of true muscle hypertrophy in contrast to other types of muscle growth such as compensatory growth (Yang and McElligot, 1989). In response to muscle hypertrophy, cross-sectional area of type II fibers have consistently been reported to increase in both rats and lambs due to β-agonist treatment (Maltin et al., 1986; Zeman et al., 1988). Along with muscle hypertrophy, β-agonists have also shown to increase the percentage of type IIA fibers while decreasing type I fibers (Vestergaard et al., 1994; Rajab et al., 2000; Bricout et al., 2004). However, anabolic agents have no effect on the distribution of type I or type IIA fibers (Ono et al., 1996; Fritsche et al., 2000). Maltin et al. (1986) conducted a study of rats fed clenbuterol for 4 or 21 d. These researchers found an increase in cross-sectional area of slow-twitch, oxidative fibers (type I), however there was no reported change in the composition of fibers. There was also an increased response to clenbuterol after four days opposed to 21 d, and there was a decrease in type IIa fibers and an increase in Type IIb fibers. In contrast, Zeman et al. (1988) conducted another experiment in which rats were fed clenbuterol. These results showed hypertrophy in fast-twitch muscles, but not in slow-twitch histochemically identified muscles. There was also a reported increase in fast-twitch to slow-twitch fiber ratio. However, in a study conducted on lambs treated for 8 wk, there was no muscle hypertrophy in Type I fibers and no change in fiber type proportion (Kim et al., 1987). In more recent years, Beerman (2002) reviewed the increase in hypertrophy of type II fibers and accounted for the increase in muscle mass without any quantitative change in muscle length. 13

22 Warner-Bratzler shear force and sensory characteristics of cattle finished on β-agonists In the meat industry, consumer acceptance is one of the most influential aspects of change. A certain level of consumer acceptability must be obtained in order for a product to have potential profits. Recent research has found tenderness to be the most important factor associated with consumers continuing to purchase a product aside from visual parameters (Savell et al., 1987, 1989; Smith et al., 1987; Miller et al., 1995). The American Meat Science Association (AMSA, 1995) published recommendations for using both sensory panels and shear force measurements in different species. Sensory panel evaluation serves as a measurement in order to rate meat products based on certain attributes such as tenderness and juiciness. Panelists are trained in order to identify certain characteristics within the meat product in order to determine if that product is more or less palatable. An objective measurement for tenderness typically used is Warner-Bratzler shear force (WBSF), which measures the amount of force needed to shear through a meat sample. Through the years, an accepted scale has been adopted in order to correlate WBSF numbers with acceptable tenderness levels. Through the work of many studies (Miller et al., 1995; Huffman et al., 1996; Boleman et al., 1997; Shackelford et al., , 1999; Wheeler et al., 1999; and Miller et al., 2001), definitive thresholds have been established in order to classify meat tenderness based on a mixture of steaks from the rib, loin, chuck, and round cooked to a medium degree of doneness. These thresholds can be categorized as the following: 1) Tender- shear force values between lbs ( kg) or less, 2) Acceptable or Intermediate- shear force values ranging from lb. ( kg), 3) Unacceptable or tough- shear force values 13 lb. or greater (>5.90 kg; Schroeder et al., 2004b). 14

23 With the use of β-agonists in cattle finishing diets, consumer approval has been studied in order to document sensory properties in meat products. Tenderness has been of most concern with the use of β-agonists not only due to its importance to consumers, but also because of initial concerns of decreased tenderness associated with the use of β-agonists. Some researchers have concluded a decrease in tenderness could be a result of increased protein synthesis (Maltin et al., 1987) or decreased protein degradation (Reeds et al., 1986). Others have found a decrease in proteolytic enzymes and an increase in the amount of proteolytic inhibitors (Kretchmar et al., 1990). This is mainly an increase in calpastatin, which inhibits the ability of the proteolytic enzymes to undergo the tenderization process in meat postmortem. Kretchmar et al. (1990) used lambs and concluded β-agonist treatment results in a decrease in proteolytic capacity in the muscle. Schroeder et al. (2004b), examined sensory properties of cattle finished on 100, 200, or 300 mg hd -1 d -1 RAC compared to the cattle on a control diet with no RAC supplementation. The authors found that no differences in sensory characteristics, including initial and sustained tenderness, for cattle finished with 100 or 200 mg hd -1 d -1 RAC. However, when the dose of RAC was increased to 300 mg hd -1 d -1, differences were seen in initial and sustained tenderness, which included a decrease in tenderness. No differences for shear force were seen for cattle treated with 100 and 200 mg hd -1 d -1 (1.58 and 1.65 kg), but when the dosage was increased to 300 mg hd -1 d -1, shear force measurements increased to 1.79 kg, which still falls within the tender threshold. According to this study, no differences were seen in tenderness measurements for cattle supplemented with RAC except for the 300 mg hd -1 d -1 RAC treatment. In a more recent article comparing the three β-agonists zilpaterol, clenbuterol, and RAC, Strydom et al. (2009) concluded that both zilpaterol and clenbuterol had increased shear force 15

24 values when compared to control diets at 2, 7, and 14 d postmortem. However, RAC was also used as a treatment in this study, and shear force values showed no differences at the specified days postmortem compared to control. This study did not perform a sensory panel to obtain subjectiv measurements. Quinn et al. (2008) completed a trial using 200 mg hd -1 d -1 Optaflexx on heifers. These results concluded that once again, shear force values for cattle treated with RAC were not different from those cattle fed the control diet. Finally, a project conducted by Avendano-Reyes et al. (2006) finished steers for the final 33 d on 60 mg hd -1 d -1 zilpaterol (Zilmax ) and 300 mg hd -1 d -1 RAC (Optaflexx ). These results found that both β-agonists had greater shear force values (zilpaterol: 5.06 vs kg; RAC: 4.75 vs kg) in comparison with control steers. Once again, it is important to note that this study fed RAC at the 300 mg level, which showed increased shear force values in this study as well as in the study conducted by Schroeder et al. (2004b). According to the literature mentioned above, studies in which RAC levels exceed 200 mg hd -1 d -1 have shown increased shear force values in beef cattle. Zilpaterol has shown increased shear force values in all studies reviewed. Meat Color Meat color is the most important factor associated with a consumer purchasing a product, because any discoloration suggests to consumers that a product is not fresh (Mancini et al., 2005). Nearly 15% of retail beef results is sold at a discounted price due to the discoloration of the meat surface, which gives rise to an annual revenue loss of over $1 billion (Smith et al., 2000). Due to the potential revenue loss from discoloration, it is important for researchers to understand the mechanism and factors associated with meat color. 16

25 Myoglobin, which is the primary protein responsible for meat color is a water-soluble protein containing eight helices (A-H) linked by short non-helical sections (Mancini et al., 2005). It consists of a protein portion known as globin and a non-protein portion, which is a heme ring. Within the heme ring is a centrally located iron which contains 6 coordination sites that bond to different elements. Iron is an element that is found in two chemical states; as the ferric (oxidized) and ferrous (reduced) forms. A determination of the opportunity for oxygen to bind myoglobin is dependent on the state that the iron atom is in. Four of the 6 coordination sites bind the iron atom to the heme ring, the 5 th site binds to the amino acid (typically histidine), and the 6 th and final site is open (ligand) to bind oxygen when present. The opportunity for the ligand to bind oxygen results in different states of myoglobin color seen in meat. Three important color pigments that the myoglobin can become are deoxymyoglobin, oxymyoglobin, and metmyoglobin. Deoxymyoglobin is the result of the iron atom of the heme ring being in the ferrous (reduced) form (Fe 2+ ; Mancini, 2005). As a result of the iron being reduced, there is no ligand present at the sixth coordination site. A purple-red meat color is the result of deoxymyoglobin, which is typical of a meat product following initial cutting. For deoxymyoglobin to maintain its color, there must be a very low oxygen tension (<1.4 mm Hg; Brooks, 1935). As meat is exposed to oxygenated environments, the color begins to take on a bright, red color. This bright, red color is known as oxymyoglobin and is the color consumer s associate with fresh meat. During oxygenation, there is no change in the valence of iron, but diatomic oxygen binds to the sixth coordination site. The stability of oxymyoglobin is dependent on many factors including ph, temperature, and oxygen competition by other respiratory processes (Mancini, 2005). Metmyoglobin is the result of a loss of electron of the iron atom, changing it from the ferrous 17

26 (Fe 2+ ) state to the ferric state (Fe 3+ ), also known as oxidation (Livingston & Brown, 1982; Wallace et al., 1982). Oxidation results in a brown color in the meat and is typical of meat that has been overwrapped in oxygen permeable film, and placed in retail display for several days. There are numerous factors associated with the formation of metmyoglobin, which include ph, oxygen partial pressure, temperature, light, and reducing activity of the meat (Mancini, 2005). Although there are subjective methods for measuring meat color, objective measurements such as L*, a*, and b* are utilized. These values are representative of the color range of black to white (L*), red to blue (a*), and yellow to green (b*), which is referred to as instrumental color (Mancini, 2005). An increase in numerical value for each of the color ranges results in an increase of lightness, redness, and yellowness (Mancini, 2005). For purposes of this project, it is important to review the affects of RAC supplementation on color stability. In a study conducted by Gonzalez et al. (2009), RAC was fed for the final 28 d of a feeding trial on beef steers. The L*, a*, and b* color measurements were observed for six muscles. In this study, RAC supplementation had no affects on color stability during retail display. In addition, a study by Quinn et al. (2008), reported that feeding 200 mg hd -1 d -1 RAC for the final 28 d of the finishing period, showed no differences for L*, a*, and b* in heifers. Lipid oxidation Although meat production has shifted to an increase in lean and a decrease in fat composition, lipids still provide important meat quality components such as flavor (Wood et al., 2004). The lipid in meat is comprised of a large number of fatty acid residues. A fatty acid is essentially made up of a long carbon chain with a carboxylic acid group on the end. Saturated fatty acids are those that contain no double bonds in the carbon chain, while chains containing one or more double bonds make up mono- and polyunsaturated fatty acids, respectively. 18

27 One of the major factors associated with meat deterioration is lipid oxidation (Fernandez et al., 1996). Meat quality is affected by lipid oxidation because it is a contributor to the development of off-flavors such as rancidity in meat products. Lipid oxidation in meat products is caused by the initiation phase in which a fatty acid is involved in a hydrolysis reaction resulting in the formation of a free radical on a carbon within a lipid (Morrissey et al., 1998). Following the formation of the radical, a triplet oxygen can react with the free radical forming peroxy-radicals (Morrissey et al., 1998). These peroxy-radicals are referred to as primary oxidation products and are not stable for a long period of time. At this point, another singlet oxygen (typically from another fatty acid) can continue to react with the peroxy-radical forming secondary oxidation products (Morrissey et al., 1998). Secondary oxidation products are typically ketones and aldehydes. Auto-oxidation continues to occur, resulting in the formation of off-flavors and the production of malonaldehyde. Malonaldehyde formation is analyzed in order to quantify the amount of lipid oxidation taking place in the product. Malonaldehyde measurements are typically analyzed using a thiobarbituric acid reactive substance (TBARS) assay. The TBA can react with the aldehyde groups that are formed in the secondary oxidation products, which gives the ability to measure the amount of oxidation taking place in the muscle. There is little research known to evaluate the effect of RAC on lipid stability in beef. A recent study conducted by Apple et al. (2008) reported that pigs fed RAC prior to slaughter and placed in retail display for 5 d showed no effect on lipid stability. This study suggests that RAC would have no effect on the lipid stability of beef steaks as well. Summary The effects of β-agonists on pigs, beef, and lambs have been thoroughly researched over the last twenty years. Due to these research efforts there is extensive evidence that β-agonists contribute 19

28 to increased average daily gain in the animal as well as serving as a repartitioning agent from fat to lean accretion in the muscle. In addition, it is also concluded that the increase in muscle size is due to muscle hypertrophy and is seen particularly in the Type II muscle fiber types. Past research has also attributed the optimum dosage of a repartitioning agent, such as RAC, will produce desirable production traits and meat composition. Based on the literature reviewed, appropriate dosage of RAC in cattle can alleviate input cost and result in increased value due to increased lean weight opposed to fat. Although there are many aspects of research in the area of β-agonists that is agreed upon by researchers, there are also some areas, such as factors associated with tenderness, that still remain inconclusive. Therefore, the objective of this experiment will be to determine the effect that RAC hydrochloride has on muscle fiber types, color attributes, and meat quality in yearling heifers across five days on feed groups. 20

29 Animals II. MATERIALS AND METHODS All experimental procedures performed at Auburn University were approved by the Auburn University Institutional Animal Care and Use Committee (AU-IACUC, PRN ). Seventy-two commercial crossbred yearling heifers were purchased from seven Alabama beef producers for this study. For 66 d prior to the experiment, heifers grazed on summer perennial pasture mixture (bermudagrass: Cynodon dactyloncl and bahiagrass: Papspalum notatum Flusge) and were fed soyhull pellets (3.2 kg hd -1 d -1 ). Following this feeding period, heifers were transported to the Auburn University Beef Cattle Evaluation facility (AUBCE) where the remainder of the experiment was completed. Animals were divided into one of five DOF groups based on initial weight and height. Days on feed groups were: 79 (n = 16), 100 (n = 16), 121 (n = 16), 142 (n = 16), or 163 (n = 7). Within DOF groups, randomly selected animals received 300 mg hd -1 d -1 Ractopamine HCl (RAC; Elanco Animal Health, Greenfield, IN) or 0 mg hd -1 d -1 RAC (CON) for the final 35 d prior to harvest. Heifers underwent a 21 d warm-up period prior to experimental feeding. At this time, animals were acclimated to individual Calan gates for feed intake measurement. During the warm-up period, heifers received 2% BW of a grain diet (Table 1) and hay mixture (bermudagrass and bahiagrass). In addition, 0.5 mg hd -1 d -1 of melengestrol acetate (MGA) was added to the diet to suppress estrus during the feeding period. Following the warm-up period, heifers had ad-libitum access to the diet fed twice a day for the remainder of the experiment. 21

30 Three heifers were removed from the growth analysis (DOF group 79, 142, 163) because they were not trainable to the Calan gates. In addition, one heifer calved (DOF group 163) on d 68 of the project and therefore was removed from the analysis as well. Following each groups allotted days on feed, heifers were humanely harvested at the Auburn University Lambert-Powell Meat Laboratory. Carcass Evaluation Following harvest, hot carcass weights (HCW) were taken for each heifer. Individual carcass evaluation was recorded on each carcass 24 h postmortem, which included USDA quality and yield grades. Carcasses were ribbed between the twelfth and thirteenth rib in order for trained carcass graders to record carcass traits for each animal (USDA, 1997). Carcass characteristics consisted of LM area; 12 th -rib fat thickness; % kidney; pelvic and heart (KPH) fat; and marbling scores. Sample Preparation Following carcass evaluation, boneless loins were removed from the right side of the carcass. Loins were vacuum-packaged and aged for 21 d at 4 ± 2 o C. Following the aging period, 7 steaks (2.54 cm) were cut from each loin starting from the anterior end. Three steaks used for a simulated retail display time (display) of 7 days for sensory analysis, Warner-Bratzler shear force, and analytical steak for TBARS analysis, respectively. Following cutting, these steaks were placed on a Styrofoam 4S size tray and overwrapped using polyvinyl chloride (PVC) film in order to go into retail display. The next 4 steaks were cut for fresh steaks for sensory panel, Warner-Bratzler shear force, analytical steak for TBARS analysis, and an extra steak, respectively. All fresh steaks were vacuum-packaged and stored in a -25 o C deep freezer until further analyses was completed. 22

31 Color Measurements Steaks for display measurements were placed in a Tyler (Model DMG-8, Niles, MI) retail display chest case with a temperature of 4 ± 2 o C. Color measurements were taken for 7 d using a Hunter Miniscan XE Plus (Hunter Laboratories Model MSXP-4500C, Reston, VA) using a 10 o observation angle, D65 illuminant and 3.5-cm aperture. Color measurements were taken on the anterior side of the steak in two locations for an average measurement of L*, a*, and b*color values. Additionally, hue angle, saturation index and the 630/580 ratio (fresh meat color) ratio were calculated during retail display time. The 630/580 ratio was calculated according to Hunt (1988), which states that 630 nm is highest peak for oxymyoglobin and 580 nm is representative of lowest peak for oxymyoglobin on an isobestic curve. Warner-Bratzler Shear Force Warner-Bratzler shear force evaluation was conducted on both fresh and 7 d displayed steaks according to the procedures by the American Meat Science Association (AMSA, 1995). Prior to cooking, steaks were thawed for 24 h at 4 ± 2 o C. After thawing, steaks were removed from vacuum packages, weighed, and placed on a preheated George Foreman clam-style grill (Model GRV120, Macon, MO). Using an Electro-Therm Digital theromometer (Model TM99A, Cooper Instruments Corp., Middlefield, CT), an internal temperature of 70 o C was achieved before each steak was removed from the grill. Steaks were then placed on a Styrofoam tray, overwrapped with PVC film, and transferred back into a refrigerator (4 ± 2 o C) in order to cool for 24 h. Following cooling, 6 cores (1.27 cm diameter) were then taken from each steak parallel to the muscle fibers. Warner-Bratzler shear force was measured on each core using a TA.XT2 Texture Analyzer (Texture Technologies Corp., Scarsdale, NY) and the mean shear force was calculated for each steak. The tests were performed using a Warner-Bratzler Probe and Guillotine set. The 23